Human Molecular Genetics, 2001, Vol. 10, No. 2 91-98
© 2001 Oxford University Press
Mouse models for the Wolf–Hirschhorn deletion syndrome
1The Jackson Laboratory, Bar Harbor, ME 04609, USA and 2MGC-Department of Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands
Received 6 September 2000; Revised and Accepted 7 November 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Wolf–Hirschhorn syndrome (WHS) is a deletion syndrome caused by segmental haploidy of chromosome 4p16.3. Its hallmark features include a ‘Greek warrior helmet’ facial appearance, mental retardation, various midline defects and seizures. The WHS critical region (WHSCR) lies between the Huntington’s disease gene, HD, and FGFR3. In mice, the homologs of these genes map to chromosome 5 in a region of conserved synteny with human 4p16.3. To derive mouse models of WHS and map genes responsible for subphenotypes of the syndrome, five mouse lines bearing radiation-induced deletions spanning the WHSCR syntenic region were generated and characterized. Similar to WHS patients, these animals were growth-retarded, were susceptible to seizures and showed midline (palate closure, tail kinks), craniofacial and ocular anomalies (colobomas, corneal opacities). Other phenotypes included cerebellar hypoplasia and a shortened cerebral cortex. Expression of WHS-like traits was variable and influenced by strain background and deletion size. These mice represent the first animal models for WHS. This collection of nested chromosomal deletions will be useful for mapping and identifying loci responsible for the various subphenotypes of WHS, and provides a paradigm for the dissection of other deletion syndromes using the mouse.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Deletion syndromes [also referred to as segmental aneusomy syndromes or contiguous gene syndromes (CGSs: the abbreviation used here)], such as Cri-du-chat (OMIM 123450), Smith-Magenis (OMIM 182290) and Williams (OMIM 194050), are an interesting class of human disease. Manifested as a consequence of hemizygosity, CGSs illustrate that diploidy is important for proper development. In the case of Prader–Willi and Angelman syndromes (OMIM 176270 and 105830, respectively), the phenomenon of genomic imprinting (parent-of-origin-dependent gene silencing) is uncovered. Thus, CGSs can offer insights into such fundamental processes as gene expression, epigenetics and regulation of development.
Two major difficulties in identifying the genes underlying component phenotypes of a CGS are that these diseases are quite rare and the deletions are usually large. In the cases of Prader-Willi, Williams and DiGeorge syndromes, the deletions often have common breakpoints, arising from recombination events catalyzed by duplicated sequences (1–3). Without randomly staggered breakpoints, it is difficult to precisely define the chromosomal regions that underlie aspects of these syndromes. Furthermore, the common ‘core’ features associated with a given CGS can vary in severity between patients, potentially confounding the mapping of syndrome components. Finally, cryptic deletions that do not have clinical consequence go undetected in humans, thereby eliminating a potential genetic mapping resource for excluding regions contributing to a CGS.
Technologies for manipulating the mouse genome have afforded opportunities to model and genetically dissect aspects of human CGSs. Individual deletions in mice have been generated to identify potential roles of genes in the Prader–Willi syndrome (4–6), and to produce heart defects characteristic of the DiGeorge syndrome (7). However, there have been no reports of using nested deletion sets to model a CGS or to dissect component phenotypes.
Wolf–Hirschhorn syndrome (WHS; OMIM 194190) is a rare CGS characterized by a ‘Greek warrior helmet’ facial appearance (prominent glabella, hypertelorism, widely set eyes), various midline closure defects, growth retardation, juvenile seizure disorders, cataracts, iris colobomas and mental retardation (8,9). Molecular characterization of deletion breakpoints in WHS patients led to the identification of a 165 kb WHS critical region (WHSCR), defined by the minimal region of deletion overlap in two key individuals. The WHSCR is located within a 2 Mb interval flanked by the Huntington’s disease (HD) and FGFR3 genes, a region sequenced during the positional cloning of HD (10). Candidate WHS genes identified in this region include WHSC1, the product of which contains motifs present in certain developmentally important proteins and exhibits an embryonic expression pattern consistent with structures affected in WHS (11), and WHSC2, a ubiquitously expressed gene of unknown function (12).
The mouse orthologs of HD and FGFR3 (Hdh and Fgfr3) map to chromosome 5 in a region of conserved synteny with human 4p16.3 (13), and thus presumably demarcate the mouse version of the WHSCR. To generate potential mouse models of WHS, a collection of deletions was induced at the Hdh locus of embryonic stem (ES) cells by irradiation (14). Mice derived from five of these lines were analyzed for phenotypes caused by segmental haploidy. Those with the larger deletions displayed several characteristics of WHS, including craniofacial dysmorphology, seizure susceptibility and midline defects. The combined data suggest that manifestation of the more severe aspects of WHS requires hemizygosity of loci outside the WHSCR, and that the traits are affected by genetic background.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In previous work, a series of nested deletions centered at the Huntington’s locus (Hdh) on chromosome 5 (14) was generated by the technique of ES cell irradiation (15). This involved three steps: (i) targeting a herpes simplex virus thymidine kinase gene (tk) into the Hdh locus of F1 hybrid (129S4/SvJae x C57BL/6J) ES cells by homologous recombination; (ii) subjecting targeted clones to ionizing radiation; and (iii) selecting for deletions of tk by culturing in medium containing 1,2'-deoxy-2'-fluoro-b-D-arabinofuranosyl-5-iodouracil (FIAU). A subset of deletion-bearing ES cell clones was injected into blastocysts to create germline chimeric mice. Five such deletions were analyzed in this study (Fig. 1). Some preliminary studies (on seizure susceptibility; see below) were performed with a deletion induced at the Dpp6 locus (Fig. 1).
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In matings to C57BL/6J (B6) females, these chimeras sired offspring that primarily inherited the non-deleted 129S4/SvJae (129) chromosome (Table 1). The few that inherited the largest deletions (namely Hdhdf2J, Hdhdf4J and Hdhdf6J) were severely growth-retarded. Transmission was increased by mating to strains C3H or CAST/Ei, yet most deletion-bearing progeny were still runted, averaging 50–80% of the weight of same-sex littermates (Fig. 2). Fitness improved on further backcrossing to C3H (data not shown). When B6(Hdhdf7J/+) N2 animals were backcrossed to B6, nearly all deletion-bearing pups were observed gasping for air immediately after birth, and died shortly thereafter. Histological examination of these pups revealed collapsed lungs (data not shown).
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Deletion breakpoints were mapped by exploiting chromosome 5 microsatellite polymorphisms between B6 and 129 in ES cells, or between either of the strains against which the deletions were placed in trans: C3H and CAST/Ei (14). To better characterize the deletions with respect to the WHSCR, experiments were conducted to ascertain the location of Whsc1, a candidate WHS gene which maps to the 165 kb WHSCR in humans (11). First, the presumed tight linkage of Whsc1 to the Hdh locus in mice was evaluated. A single strand conformation polymorphism (SSCP) was used to genetically map Whsc1 on the Jackson Laboratory’s BSS interspecific backcross mapping panel (http://www.jax.org/resources/documents/cmdata/ ). Whsc1 was inseparable from the D5Mit148 and D5Mit149 loci that flank Hdh. Second, a restriction site polymorphism between strains C3H and B6 in the 3'-untranslated region of Whsc1 was identified and exploited to screen the deletion set. Whsc1 was absent in all deletions.
To better characterize Hdhdf9J, which retained heterozygosity at all polymorphic microsatellites near Hdh, FISH analysis of ES cells was performed using mouse PACs corresponding to the WHSCR syntenic region as probes (Fig. 3E). This revealed that cells bearing the Hdhdf9J deletion are hemizygous for all genes in the LetM1–Hdh interval, which spans 
2 Mb in humans (the physical size of this region in mice is not known), but retained two copies of the closest flanking microsatellite markers (D5Mit149 and D5Mit388) which lie within 1 cM of Hdh according to the Mouse Genome Database. Thus, Hdhdf9J, as well as all the other Hdh deletions used in this study, span the entire 165 kb WHSCR of humans (Fig. 1).
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Since hemizygosity of the WHSCR is the molecular basis for diagnosis of WHS in humans, we examined deletion-bearing mice for WHS-like phenotypes. Several animals harboring the deletions Hdhdf2J, Hdhdf4J, Hdhdf6J and Hdhdf7J in a background containing >50% contribution from B6 showed craniofacial features potentially related to the hallmark ‘Greek warrior helmet’ phenotype of WHS (Fig. 3A). The most prominent anomalies were a domed skull (WHS patients generally have a protruding nasal bridge), smaller ears (lobeless ears in WHS), widely set eyes (typical of WHS) and, in the most severe cases, a marked facial asymmetry (also typical of WHS) (Fig. 3B and C). Autopsies of two such animals revealed maloccluded lower incisors (Fig. 3B), misshapened palatine ridges (Fig. 3B), asymmetry of the jaw bones (data not shown) and tail kinks (Fig. 3C), suggesting analogy to midline defects in WHS patients. Tail kinks were commonly present in mice containing the Hdhdf2J, Hdhdf4Jand Hdhdf6J deletions, especially in markedly runted animals.
The penetrance and expressivity of marked craniofacial anomalies was clearly background dependent, being most pronounced in the B6 strain. Attempts to quantitate penetrance was confounded by the lethality that occurred in crosses of the largest deletions to this strain (described above). Nevertheless, in cases of individual affected animals that produced multiple deletion-bearing offspring in crosses to B6, the character was reliably transmitted. For example, all the deletion-bearing offspring (4 of 21 progeny) of one particular Hdhdf4J male mated to B6 had typical craniofacial defects (domed skull, short ears, wide eyes as in Fig. 3A). However, when the same male was outcrossed to a non-inbred stock, the deletion-bearing offspring (6/11) had subtle or no observable craniofacial defects (the phenotype is somewhat subjective). To investigate the ontogeny of the craniofacial abnormalities, a litter produced by a mating of this male to a B6 female was sacrificed at birth, and bone/cartilage preparations of the skulls were prepared. There were no clear defects observable in the skulls of the four deletion-containing pups in this litter of 10 (data not shown), suggesting that the craniofacial dysmorphologies develop during postnatal growth in the mouse.
Two studies found that >85% of WHS patients presented with seizure disorders (16,17). In the course of normal mouse husbandry (cage changing; weaning), we observed several episodes of spontaneous seizures displayed by deletion-bearing mice. To systematically gauge seizure susceptibility, we tested animals for incidence of electroshock-induced clonic seizures. As detailed in Table 2, only 12% (4/34) of wild-type control littermates suffered induced seizures, whereas >85% of the mice heterozygous for Hdhdf6J and Hdhdf7J seized in response to the stimulus. The seizure incidence for each deletion was significantly higher than littermates in both cases (Table 2) and, in general, were qualitatively more severe and sometimes terminal (i.e. maximal tonic-hindlimb seizures). Hdhdf4J heterozygotes also had more seizures than littermates (60 versus 11%, respectively), but the difference was not significant with the number of animals tested. Notably, half of the seizures induced in Hdhdf4J/+ animals were maximal, but no maximal seizures were observed in controls. The induced seizure incidence in Hdhdf9J/+ mice was similar to that in controls, although occasional Hdhdf9J/+ animals were observed to suffer spontaneous convulsive seizure episodes. It is conceivable that deletion of WHSCR alone may be insufficient to confer electroshock-induced seizure susceptibility, but that spontaneous seizures might represent a distinct phenotype. It is also possible, in light of the lower seizure frequency observed in Hdhdf4J/+ mice, that multiple regions contribute to seizure susceptibility. These issues can be more rigorously addressed if the deletions can be rendered congenic on a defined strain background(s). To determine whether hemizygosity of regions flanking, but not including, the WHSCR cause seizure susceptibility, mice heterozygous for a deletion centered at the Dpp6 locus, Dpp6df1J, were tested by electroshock. This deletion overlaps Hdhdf4J over an 5 cM stretch between D5Mit348 and D5Mit148 (Fig. 1). None of the four animals tested had an induced seizure (Table 2).
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Colobomas (midline closure defects of the optical stalk and/or cup) and cataracts are typical components of the WHS phenotype (18). Several Hdhdf4J/+ and Hdhdf7J/+ animals exhibited corneal opacities (Fig. 3D). Slit lamp biomicroscopy revealed eye defects in 23% (9/38) of the Hdhdf7J/+ mice examined: four animals had iris colobomas (Fig. 3D), four had cataracts and one had both. No defects were found in Hdhdf9J/+ mice (0/27) of comparable genetic background (
50% B6). Sporadic colobomas were also observed in seven Hdhdf4J/+ mice and two Hdhdf6J/+ animals. Interestingly, no corneal opacities have been observed in mice bearing the Hdhdf2J deletion, which is larger than both Hdhdf4J and Hdhdf6J (insufficient animals were examined for colobomas to draw conclusions). One of the most defining hallmarks of WHS is mental retardation. In a first attempt to identify possible learning deficits, mice bearing the largest deletions were subjected to the Morris Water Maze test. No differences were noted in comparison with controls (data not shown). However, gross examination of brains from mice heterozygous for Hdhdf2J, Hdhdf4J, Hdhdf7J and Hdhdf9J revealed that the colliculi of mice bearing deletions were more exposed than those of wild-type controls due to hypoplasia of the cerebral cortex (Fig. 4, arrows). It is unknown whether this anatomical defect has any functional consequences.
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Although mice bearing the Hdhdf2J and Hdh4J deletions scored normally in the water maze, we noted that they swam unusually. Furthermore, they displayed an unusual ‘froglike’ gait, in which their hindlimbs splayed outward from the body axis as they walked. To investigate, whole mount and sagittal sections through the cerebella of mutant and control animals were examined. Lobulation defects as a reduction in cerebellar size was seen in mice heterozygous for Hdhdf4J and Hdhdf2J. Whether these defects underlie the abnormal gait is unclear. No marked cerebellar defects were seen in Hdhdf7J and Hfhdf9J mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have shown in this report that mice carrying large deletions encompassing the Hdh locus showed several phenotypes characteristic of WHS. These included seizure susceptibility, iris colobomas, cataracts/corneal opacities, craniofacial dismorphology, midline defects and growth retardation. It must be cautioned that growth retardation is a common phenotype associated with chromosomal deletions in mice (19) and most other human CGSs. Thus, it is not clear whether the basis for runting in the Hdh deletion mice is identical to that which underlies intrauterine growth retardation of WHS patients.
Neonatal mortality, which has also been associated with WHS (18), was especially marked on breeding to the B6 genetic background. Interestingly, a similar phenotype was reported in 129,B6(Ndntm1Stw/+) mice, a model for Prader–Willi syndrome (20). It is possible that strain B6 is intolerant of haploid gene expression at these loci or causes certain hemizygous genes to be expressed at subhaploid levels. Another possibility is that, in the B6 background, some genes in deletion regions might undergo randomized uniparental inactivation, causing a subset of cells to be effectively nullizygous. Finally, the phenotypic severity of deletion-bearing animals might be related to the genotype of the alleles present on the non-deleted chromosome. These hypotheses can be experimentally addressed in the mouse system.
One of the more intriguing findings presented here was that mice heterozygous for the Hdhdf9J microdeletion, which removes the mouse version of the WHSCR but minimal flanking DNA, did not exhibit the prominent WHS-like characters. There are several possible explanations. One is that the WHS-like phenotypes associated with the larger deletions are completely unrelated to the genetic causes of WHS in humans. This possibility would be difficult to prove or disprove. Another explanation is that hemizygosity of the WHSCR alone may be necessary but not sufficient for the full manifestation of WHS. Since there are no known WHS patients who delete only the WHSCR or subregions thereof, it is currently impossible to evaluate this possibility. However, this hypothesis can be tested in mice. If true, then one prediction would be that mice containing deletions that remove DNA flanking, but not including the WHSCR, would fail to display the WHS phenotypes reported here. Since mice with the recently isolated Dpp6df1J deletion did not exhibit seizure susceptibility or other overt WHS-like defects (data not shown), WHSCR-extrinsic genes causing potential autonomous WHS phenocopies would have to reside between D5Mit388 and Fgfr3 (the region deleted by Hdhdf2J proximal to the WHSCR) and/or Add1 and D5Mit421 [the region deleted by Hdhdf4J and Hdh7J distal to the WHSCR (Fig. 1)]. We are currently in the process of analyzing additional deletions centered at Dpp6 (14) and inducing deletions centered at Qdpr. Analyses of such mice will allow refinement of chromosomal regions containing haploinsufficent genes underlying the phenotypes reported here.
A variation on the hypothesis that both hemizygosity of the WHSCR and deletion of flanking regions is needed to manifest the core phenotypes of WHS (Greek warrior helmet, seizures, mental retardation) posits that additional phenotypes associated with the syndrome, or modifiers of the syndrome’s severity, might be related to the extent of DNA deleted flanking the WHSCR. In support of this view, a correlation between deletion size and clinical severity has been documented for WHS patients in multiple studies (17,21,22). Furthermore, it must be considered that the WHSCR was defined on the basis of two patients (CM and LGL7447) who had deletions (of 2.5 and >4 Mb) extending in opposite directions, overlapping only in the WHSCR (10,23). Shared features of these patients were seizures, abnormal facies and developmental delay. However, they were not reported to have other defects typical of WHS, such as colobomas, cleft palate and heart defects. Indeed, analysis of a cadre of patients with chromosome 4p deletions indicated that some anomalies typical of WHS can be attributable to regions outside the WHSCR (24). In contrast, it was found that certain patients with Pitt–Rogers–Danks syndrome, which is characterized by milder WHS-like phenotypes, not only overlap WHS deletions but in some cases delete larger stretches of DNA (25,26).
It is evident from examples such as these and others in the literature that dissecting the genetic basis of phenotypes associated with WHS, using exclusively clinical data, will be exceedingly difficult or impossible for the following reasons. First, WHS is quite rare, precluding sufficient redundancy by which to draw confident conclusions on the phenotypes that can be ascribed to a particular chromosomal interval. Second, the variability in the severity and spectrum of abnormalities associated with 4p deletions complicates and exacerbates this problem. Third, since subclinical individuals carrying microdeletions would be identified only rarely, it is difficult to rule out particular regions from being involved in WHS.
The mouse provides a model system that is suited to overcome the difficulties posed by a disease like WHS. The ability to manipulate the mouse genome in many ways can be exploited to systematically address the genetic basis of WHS. The results presented here lay the groundwork for such studies. Not only do the deletion-bearing mice appear to represent relevant models of WHS, but the severity of the phenotypes is highly influenced by genetic background, as appears to be the case in humans. Unlike human patients, the ability to generate an unlimited number of mice with an identical modification permits more robust and informative analysis of the consequences of particular genetic lesions.
Our collection of nested deletion mice, plus others that can be generated from cryopreserved, deletion-containing ES cell clones centered not only at Hdh but also at flanking loci (14), will be valuable for mapping loci involved in the WHS-like defects. The current data allow us to assign candidate regions for some of the phenotypes examined (the phenotypes of all deletions are summarized in Table 3). The cerebellar lobulation defect can be localized to either of two intervals: (i) between D5Mit389 and D5Mit388, defined by the proximal breakpoints of Hdhdf2J, which has the defects, and Hdhdf7J, which does not; or (ii) between D5Mit351 and D5Mit421, in the event that the distal breakpoint of Hdhdf4J (which has the cerebellar defects) extends more distal to that of Hdhdf7J. The seizure phenotype lies in a region maximally defined by the Hdhdf7J deletion (unless the distal breakpoint of Hdhdf4J is more proximal), but which excludes the region deleted in Hdhdf9J. The coloboma phenotype can be tentatively localized to the interval between the proximal breakpoint of Hdhdf6J (D5Mit388) and the distal breakpoints (D5Mit421) of Hdhdf7J or Hdhdf4J, all of which have the defect, but not within the region deleted by Hdhdf9J. The failure to observe corneal opacities in Hdhdf2J mice is puzzling. It is possible that this deletion is discontinuous, such that an island of DNA containing the locus responsible for this haploinsufficient defect remains present. Alternatively, this observation may be related to the incomplete penetrance of the corneal opacities, which are likely due in part to genetic background effects. The Hdhdf2J deletion could not be transmitted in crosses to C57BL/6J animals (Table 1). It is possible that those background alleles which predispose to corneal opacities are incompatible with viability of Hdhdf2J/+ mice.
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As regions containing these loci are narrowed, complementation with individual candidate transgenes or large-insert genomic clones can be attempted to ultimately identify those that are responsible for phenotypes. Alternatively, knockouts of individual WHS candidate genes such as Whsc1 can be generated, but the results here suggest that mice with heterozygous deficiencies of individual genes in the WHSCR will not reproduce the full clinical spectrum of the disease. Nevertheless, such knockouts might be combined with flanking deletions to test the idea that hemizygosity of genes both within and outside the WHSCR might be required for phenotypic manifestation. Finally, further characterization of genetic background effects may lead to the identification of loci that participate in the developmental processes that are affected in WHS.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Induction and characterization of deletions
Targeting into the Hdh gene (C57BL/6J allele), induction of deletions in ES cells by irradiation and characterization of several hundred deletion clones with microsatellite markers have been described elsewhere (14). Molecular detection of the Whsc1 locus was assessed by PCR, using primers (whs1, 5'-ATCTAAAATAGAAATGGGAGGG-3'; whs2, 5'-CCAGGATGGGCAGTCC-3') specific for the 3'-untranslated region, followed by digestion of the products (
445 bp) with TfiI. Whereas the B6 and 129 alleles of Whsc1 contain two TfiI sites, C3H has one.
Tests for seizure susceptibility
Susceptibility to minimal clonic seizures (27) was assessed by a high frequency electroconvulsive threshold test. Briefly, animals were restrained by hand, a drop of saline solution containing the topical anesthetic tetracaine (0.5%) was placed on each eye and 7 mA current was applied via silver transcorneal electrodes using a electroconvulsive stimulator (Ugo Basile model 7801). The stimulator was set to produce rectangular wave pulses with the following properties: 299 Hz, 0.2 s duration, 1.6 ms pulse width. Seven milliamperes is subthreshold for C57BL/6J mice: the CC50 (critical current for 50% to have a minimal seizure) in females = 7.92 mA (95% CI: 7.67–8.29); males = 9.35 mA (95% CI: 9.11–9.69).
Eye examinations
A Haag-Streit 900 slitlamp set at a magnification of 40x was used for ocular examinations. After dilation of the pupil with 1% cyclopentolate, the fundus was examined using a binocular indirect ophthalmoscope.
FISH analysis
ES cells treated with 0.1 M KCl were fixed with 3:1 methanol:acetic acid, dropped on glass slides and air-dried overnight. PACs were labeled by nick translation using either biotin-11-dUTP or digoxigenin-11-dUTP. Probes were pre-annealed for 1–2 h in the presence of 2 µg of mouse Cot1 DNA, then hybridized to the slides overnight. Dual-color fluorescence techniques were performed essentially as described (28). Signals were visualized in three detection steps using either Streptavidin Alexa594 (1:100; Molecular Probes), goat anti-StreptavidinBio (1:100; Vector) and Streptavidine Alexa594 (1:100) or mouse anti-DIG–FITC (1:250; Sigma), rabbit anti-mouse FITC (1:1000; Sigma) and goat anti-rabbit FITC (1:1000). The slides were mounted in Vectashield (Vector) containing DAPI (100 ng/ml; Roche).
Histology and whole brain mounts
Adult mice were deeply anesthetized with avertin and transcardially perfused with Bouin’s solution. Skulls were postfixed overnight and brains were removed for examination under a dissecting stereomicroscope (Wild M10; Leica) prior to paraffin embedding. Brains were sectioned sagittally and 7 µm sections were stained with hematoxylin and eosin. Sections were examined using a Leica DMRXE microscope. High resolution digital images were captured by a Spot 1.1 camera (Diagnostic Instruments). The samples shown in Figure 4 are representative of the following numbers of individuals examined: two Hdhdf2J, three Hdhdf4J, one Hdhdf7, two Hdhdf9J and three control (non-deletion) littermates.
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ACKNOWLEDGEMENTS |
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We are grateful to M. MacDonald for Hdh plasmids and phage clones; K. Hirschhorn, M. Altherr, T. Vogt, M. Bucan, M. MacDonald, S. White and the members of the Schimenti laboratory for valuable advice and discussion; T. Gridley, J. Naggert and J. den Dunnen for critical reviews of the manuscript; A. Costa and K. Walsh for conducting the Morris water maze tests; E. Gutteling for technical assistance with FISH; C. Mooser for technical assistance; and Johan den Dunnen for continuous support of A.V. This work was supported by NIH grant HD35984 to J.C.S., NIH grants NS31348 and NS40246 to W.N.F., Cancer Center grant CA34196 to the Jackson Laboratory, and the Leiden University Medical Center. N.C.G. was supported by a training grant in Developmental Biology, HD07065.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 207 288 6402; Fax: +1 207 288 6082; Email: jcs@jax.org
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REFERENCES |
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